TIBTEC 1886 No.

of Pages 12

Trends in Biotechnology

Opinion

Intraoperative Bioprinting: Repairing Tissues

and Organs in a Surgical Setting
Yang Wu,1,2,3 Dino J. Ravnic,4 and Ibrahim T. Ozbolat2,3,5,6,*

3D bioprinting directly into injured sites in a surgical setting, intraoperative Highlights

bioprinting (IOB), is an effective process, in which the defect information can Recent progress in bioprinting has
be rapidly acquired and then repaired via bioprinting on a live subject. In patients advanced beyond in vitro applications
toward intraoperative bioprinting.
needing tissue resection, debridement, traumatic reconstruction, or fracture
repair, the ability to scan and bioprint immediately following surgical preparation Different intraoperative bioprinting mo-
of the defect site has great potential to improve the precision and efficiency of dalities possess pros and cons in spe-
these procedures. In this opinion article, we provide the reader with current cific biomedical applications.
major limitations of IOB from engineering and clinical points of view, as well as
The advantages of intraoperative
possibilities of future translation of bioprinting technologies from bench to bioprinting bring forth opportunities to
bedside, and expound our perspectives in the context of IOB of composite and clinically translate bioprinting technolo-
vascularized tissues. gies from bench to bedside.

Intraoperative bioprinting of composite

tissues has drawn particular interest
Advances from in Vitro Bioprinting to Intraoperative Bioprinting since the repair of natural defects usually
In the last decade, bioprinting has considerably advanced and a large number of studies have entails the reconstruction of multiple dif-
shown attractive outcomes in different applications, such as engineering of various organs for ferent tissue types.
regenerative medicine (i.e., skin [1], cartilage [2], and bone [3]), tissue vascularization [4], drug
discovery [5], and disease modeling [6]. However, the majority of bioprinting endeavors have
been conducted in vitro and, in some cases, further validated in vivo [7]. Although bioprinting of
solid organs directly into human body remains elusive, some attempts have been practiced in
the realm of intraoperative bioprinting (IOB) (see Glossary), also known as in situ or in vivo
bioprinting [8–10], which refers to the bioprinting process performed on a live subject in a surgical
setting, in which defect imaging, data processing, process planning, and bioprinting are per- 1
School of Mechanical Engineering and
formed consecutively in a single process. IOB of tissue substitutes directly into injury sites is Automation, Harbin Institute of
Technology (Shenzhen), Shenzhen
greatly beneficial as it can facilitate the complex tissue heterogeneity in an anatomically accurate
518055, China
manner. Although IOB has not been performed in clinics yet, the field is steadily moving forward 2
Engineering Science and Mechanics
owing to the rapid developments in bioprinting technologies with contributions from interdisciplin- Department, The Pennsylvania State
University, State College, PA 16801,
ary teams of researchers from different domains spanning from engineering and materials to
USA
medical sciences and surgery. 3
The Huck Institutes of the Life Sciences,
The Pennsylvania State University, State
College, PA 16801, USA
The realization of IOB will bring about several benefits. First, bioprinted tissue substitutes, espe- 4
Department of Surgery, Penn State
cially hydrogel- or cell aggregate-based constructs, usually have weak initial mechanical Health Milton S. Hershey Medical Center,
strengths due to the fluid-rich nature, which makes them difficult to handle with surgical tools Hershey, PA 17033, USA
5
Department of Biomedical Engineering,
and further increases the likelihood of construct disintegration while being transferred to the
Penn State University, University Park, PA
surgical site and suturing. In contrast, with the assistance of a reverse engineering methodology 16801, USA
6
enabling the real-time design of the graft, IOB is an effective process, where the defect can be Materials Research Institute, Penn State
University, University Park, PA 16801,
repaired with minimum risk of contamination, graft disintegration, and manual interventions
USA
such as stacking multiple layers, in vitro culturing bioprinted scaffolds, transportation during sur-
gery, or modifying prefabricated scaffolds conforming the defect shape [11]. More importantly,
IOB is able to tackle natural defects with irregular topographies, which is common in clinical *Correspondence:
cases due to trauma and surgical excision. Comparatively, in vitro bioprinting usually assumes ito1@psu.edu (I.T. Ozbolat).

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© 2020 Elsevier Ltd. All rights reserved.
Trends in Biotechnology

a flat working surface, which is inconsistent with clinical scenarios. Moreover, since intraopera- Glossary
tively repaired defects are surrounded by native tissue, endogenous cells could be directed by Arteriovenous loop: an anastomosis
proper biochemical and biophysical cues to migrate into the bioprinted constructs and differen- between an artery and a vein, where
tiate into target tissue-specific lineages [11,12]. In addition, compared with manual injection of arterial blood is pumped into the vein.
Artificial intelligence: a field of science
biomaterials into defect sites, IOB enables the precise deposition of cells, genes, or cytokines
and engineering concerned with the
with localized control and anatomical biomimicry. For tissues that are heterocellular and formed computational understanding of
of zonally stratified arrangement of extracellular matrix (ECM), IOB is a powerful technology intelligent behavior and with the creation
to precisely reconstitute multiple layers that is quite challenging using manual approaches. Lastly, of artifacts that exhibit such behavior.
Craniomaxillofacial (CMF) defects:
shape distortion of hydrogels (e.g., high degree of shrinkage of collagen during skin regeneration major facial defects where hard and soft
[13]) can be an issue during in vitro maturation of bioprinted constructs. In addition, during in vitro tissues are rendered abnormal,
culture, cells remodel the matrix and eventually change the shape of the bioprinted constructs, dysfunctional, or deformed due to
which may pose a significant disruption of predesigned shapes. Therefore, IOB is also advanta- trauma, resection of a tumor, or the
result of a disease process.
geous due to the concurrence of in vivo integration, where the regenerated tissue may occupy the 3D photogrammetry: the process of
void space due to shrinkage and regulate the remodeling of matrix. getting shape measurements and
extracting 3D information from
photographs. Digital photogrammetry
Current Developments in IOB
operates on images of an object
Although the concept of IOB was first proposed in 2007 [14], very few studies have been reported captured from different locations and
on this topic, as bioprinting on a live subject is not trivial. Indeed, IOB is more demanding with angles using digital cameras and has
respect to bioink preparation, bioprinter setup, sterilization, and surgical operation as compared computer-detected overlapping
patterns to 3D reconstruct the
with implanting prefabricated constructs. Thus far, some attempts targeting the repair of carti-
photographed model.
lage, bone, and skin defects have been reported by different groups. For example, handheld de- Extracellular matrix (ECM): the
vices have been developed for cartilage repair in a sheep model [15,16] (Figure 1A) and skin repair noncellular portion of a tissue, which is
in murine and porcine models [17]. Although the deposition was performed manually, it has come produced and secreted by cells. The
main function of ECM is to provide
up with a well-developed strategy to miniaturize the number of external equipment for
structural and biochemical support to
crosslinking of a cell-laden hydrogel. In terms of bone repair, Keriquel and coworkers [18] have surrounding cells.
performed IOB in the mouse calvaria defect model using an in-house developed biological Inosculation: in plastic surgery,
laser-based bioprinting (LBB) system (Figure 1B). In their follow-up studies, IOB gave rise to inosculation means that blood vessels
from the recipient site connect with
organized microvascular networks and bone regeneration in calvaria defects [19,20]. Currently, those of the graft in order to facilitate
the most successful cases on IOB are mostly associated with skin repair due to its ease of access subsequent blood perfusion and restore
and regenerative potential [21,22]. Most recently, Albanna and colleagues presented a mobile vascularity.
skin bioprinting system with integrated imaging technology for on-site management of murine Intraoperative bioprinting (IOB): a
bioprinting process that is performed on
and porcine full-thickness skin wound models [23] (Figure 1C). Table 1 gives a summary of the a live subject during the course of a
past works performed in IOB and their significant outcomes. A few excellent review articles surgical operation, in which defect
provide more detailed information on the current status of in situ bioprinting [8–10]. Beyond the imaging, data processing, process
planning, and bioprinting are performed
prior reviews, here we focus on the current major limitations of IOB from engineering and clinical
consecutively in a single process.
points of view, highlighting the potential of future translation of IOB technologies, and also Miniaturization: to manufacture ever
expounding the possibilities of using such a technology in reconstruction of composite and smaller mechanical, optical, and
vascularized tissues. electronic devices.
Oxygen-generating biomaterials
(OGBs): OGBs are designed to provide
Considerations and Future Outlook for IOB a gradual and prolonged oxygen supply
Limitations of Current Bioprinting Modalities to cells as rapid or high concentration of
Bioprinting modalities can be categorized as extrusion-, droplet-, and laser-based, which oxygen potentially causes cell death due
to the formation of free radicals.
have been comprehensively reviewed elsewhere [24–26] and are also summarized in Box 1.
Path planning: generation of a path for
For IOB, more challenges appear when adapting knowledge gained through in vitro systems. a print head to deposit bioink to fill the
Extrusion-based bioprinting (EBB) can be considered an appropriate modality for IOB since defect with designated pattern based on
manual injection (extrusion) of biomaterials has already been clinically applied for decades the scanned 3D model.
[27,28] and current surgical robots, used in some clinical applications such as urology and Stem cells: one of the human body's
master cells, which are unspecialized
gynecology [29], can be easily reconfigured to hold EBB tips. However, the print tip might in-
(undifferentiated) and retain the ability to
terfere with defect periphery since it is a contact-based technology. In terms of droplet-based divide throughout life and give rise to
bioprinting (DBB), although its resolution is superior than that of EBB, bioink deposition cells that can become highly specialized

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Trends in Biotechnology

through a small orifice results in higher risk of nozzle clogging, which may increase the dura- and take the place of cells that die or are
lost. Popular stem cells in bioprinting
tion of surgery and may bring other complications. Limited by poor structural and mechanical include mesenchymal stem cells
integrity of bioprinted constructs, DBB is more suitable for bioprinting thin tissues such as (MSCs), induced pluripotent stem cells
skin. For LBB, it shares similar advantages (e.g., high resolution) and disadvantages (iPSCs), adipose-derived stem cells
(e.g., low integrity of bioprinted constructs) with respect to DBB. The miniaturization of (ADSCs), etc.
Vacuum assisted closure: a type of
LBB is a major challenge due to its complex setup (such as a laser source and optical com- therapy to help wounds heal. During the
ponents), which constrains its accessibility to internal tissues. Other challenges, such as the treatment, a device decreases air
immobilization of light sources and the necessity of precise focusing of light, may limit its pressure on the wound, which can help
clinical translation. the wound heal more quickly.

Trends in Biotechnology

Figure 1. Examples for Intraoperative Bioprinting (IOB). (A) IOB using the biopen for treatment of a full-thickness chondral defect in a sheep; adapted, with
permission, from [16]. (B) Laser-based IOB for skull repair in a mouse calvarial defect; adapted, with permission, from [18–20]. (C) Droplet-based IOB for skin repair in a
porcine model; adapted, with permission, from [23].

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4

Table 1. Summary of Studies Performed in the Context of IOB

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Bioprinting Materials Cell source Animal Defect Target 3D Results Refs

modality
Extrusion-based Gelatin methacrylamide
bioprinting (GelMA)/hyaluronic acid
methacrylate (HAMA)
Alginate/fibrin/collagen/
hyaluronic acid (HA)
Laser-based Nano-hydroxyapatite
bioprinting (n-HA)
Collagen/n-HA
Collagen/vascular endothelial
growth factor (VEGF)
type model tissue scanning
Mesenchymal stem Sheep Chondral Cartilage No • Better overall macroscopic appearance in the [16]
cells (MSCs) defect handheld printed group compared with control
groups
• Handheld printed constructs showed a higher
amount of newly regenerated cartilage and the
absence of subchondral bone deformation or
collapse
• Handheld printed constructs showed positive
Safranin O and collagen type II staining
Fibroblasts/ Mice/pig Skin Skin No Murine model: [17]
keratinocytes defect on • In situ deposition of an architected sheet was
dorsa performed in the form of a fiber array onto a small
and compliant wound surface
Porcine model:
• The porcine model demonstrated successful
in situ bioprinting to cover full-thickness wounds
with a hemostatic barrier where it did not impede
normal re-epithelialization or wound contraction
• Microscopic analysis of healed wounds on day
20 revealed that both treated and control
wounds formed complete granulation tissue and
exhibited comparable levels of collagen
deposition and cellularity.
– Mice Calvaria Bone No • Material was present in close contact with [18]
defect dura mater on the test sites after 1 week
• After 1 month, newly formed mature and
immature bones and n-HA aggregates inside
macrophages were observed
• Three months after printing, mature bone
tissue was observed
• From 1 week to 3 months, the amount of
n-HA particles observed in situ decreased
MSCs Mice Calvaria Bone No • Both n-HA-collagen and n-HA-collagen + cells, [19]
defect printed in a ring geometry, show only a marginal
tissue reconstruction at 2 months postprinting,
particularly from the periphery of the defect
• The n-HA-collagen + cells, printed in disc
geometry, show a substantial new bone
formation, well distributed throughout the defect,
even in the center of the defect
Stem cells from the apical Mice Calvaria Bone No • Randomly seeded cells were poorly organized [20]
papilla (SCAPs)/human defect within the region of interest in the defect at
umbilical vein endothelial 2 months
cells (HUVECs) • For the ‘ring’ condition, the geometry of
vascularized area was consistent with the
initial printed pattern. For the ‘disc’ and
‘crossed circle’ patterns, the initial pattern
was distorted
 Trends in Biotechnology
• Vascularization rate and bone regeneration rate
significantly increased in the ‘disc’ or ‘crossed
circle’ patterns of HUVECs
• HUVECs are not the only cells involved in the
network formation and host murine cells seem to
have a role in this process
Droplet-based Fibrin/collagen Amniotic fluid-derived Mice Skin Skin No • AFS cell- and MSC-driven wound closure and [21]
bioprinting stem cells wound re-epithelialization were significantly greater than
(AFSCs)/MSCs/HUVECs on dorsa wounds treated by fibrin-collagen only
• Microvessel density and capillary diameters in
AFS cell-treated wounds increased compared
with MSC-treated wounds, whereas the skin
treated only with gel showed the least
microvessels
• AFS cells secreted growth factors at higher
concentrations than MSCs, resulting in increased
wound closure rates and angiogenesis
HA/heparin-conjugated AFSCs Mice Skin Skin No • Deposition of the HA-HP + AFS cells [22]
hyaluronic acid (HA-HP) Wound accelerated closure of wounds faster than
on dorsa HA-only hydrogels and treatment-free controls
and induced increased vascularization
• The increased levels of elastin, GAGs, and
proteoglycans, and decreased relative expression
of collagen type I, suggested the regenerated
skin using HA-HP + AFS cells is more pliable and
elastic compared with control groups
Fibrin/collagen Fibroblasts/keratinocytes Mice/pig Skin Skin Yes Murine model: [23]
defect on • Printed skin cells closed the wound by 3 weeks
dorsa postsurgery compared with 5 weeks for
untreated and matrix-only groups
• Epithelialization of bioprinted wounds was
observed, forming an immature epidermal barrier
Porcine model:
• Bioprinting of autologous cells resulted in
~3-week acceleration in wound closure
compared with other treatments (i.e., untreated,
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matrix, and allogenic cell-treated)

• Bioprinting autologous cells resulted in ~50%
reduction in wound contraction compared with
other treatments
• Bioprinting autologous cells resulted in a 4–
5-week acceleration in wound re-epithelialization
compared with other treatments
• Collagen fibers present in the autologous cell-
treated wounds appeared larger and more orga-
nized than other groups
Comparison with cell spraying:
• Bioprinted wounds showed accelerated forma-
tion of epidermis and more mature dermis tissue
• Staining of collagen fibers were more prominent
in the bioprinted wounds at week 4 compared
with wounds treated with the sprayed cells
5
Trends in Biotechnology

Box 1. Comparison of Bioprinting Modalities Regarding Intraoperative Bioprinting

Extrusion-based bioprinting: Bioprinting of a bioink solution using an extrusion mechanism, resulting in deposition of cells laden in the bioink in the form of cylindrical
filaments of customized 3D structures.

Strengths

• Compatible with a large variety of bioink materials

• High mechanical strength with structural integrity, suitable for reconstruction of hard tissues
• Appropriate modality for intraoperative bioprinting due to its similarity to manual injection, which has been clinically applied
• Enables bioprinting of scaffold-free bioinks such as tissue spheroids
• Commercially available with moderate cost.
Limitations

• Print tip might interfere with defect periphery due to its contact-based mechanism
• Cell damage due to high shear stress
• Low resolution preventing bioprinting of thin layers of tissues, such as the skin, with a stratified arrangement of multiple layers.
Droplet-based bioprinting: Bioprinting of a bioink solution with a droplet deposition mechanism medicated by electrical, thermal, or acoustic energy.
Strengths

• Capable of bioprinting multiple types of cell

• High resolution with high-throughput capability
• Affordable, versatile, and commercially available
• Non-contact bioprinting (the nozzle does not interfere with the defect).
Limitations

• Compatibility with low viscosity bioinks (in the range of 3.5–12 mPa s)
• Poor structural and mechanical integrity of bioprinted constructs
• Small orifice results in higher risk of nozzle clogging, which may increase the duration of surgery and associated risks.
Laser-based bioprinting: Bioprinting of bioink with the laser energy as the major deposition mechanism, allowing high-precision patterning of biologics or
fabrication of tissue constructs.
Strengths

• Compatibility with viscosities in the range of 1–300 mPa s

• High resolution with the capability of single cell printing
• Nozzle-free bioprinting resulting in negligible cell damage
• Non-contact bioprinting.
Limitations

• Poor structural and mechanical integrity of bioprinted constructs

• Complicated to operate and difficult to miniaturize for a surgical setting
• Labor intensive and time consuming leading to increased surgery duration
• Higher cost and no commercial availability.

Compatibility of Bioinks to Surgical Settings

Since the deposition is performed directly into a defect in physiological conditions, an ideal bioink
should not only meet the general bioink requirements [30] but also possess some features spe-
cific to IOB. In particular, such bioinks are expected to be: (i) compatible with and intraoperatively
bioprintable by the targeted modality to shorten the surgery duration, (ii) rapidly crosslinkable
in situ to retain the integrity and resolution of bioprinted constructs in physiological temperature
and moist environment, and (iii) commercially available and affordable to minimize the surgery
cost. Therefore, most of the popular biomaterials (e.g., polycaprolactone, polylactic acid, etc.),
which rely on volatile organic solvent and high melting temperature, become inappropriate.
Among hydrogels, although collagen transitions to gel state at 37°C, its slow gelation hinders
its use in IOB; however, nano-hydroxyapatite-reinforced collagen has shown promising results
in bone regeneration [19]. Fibrinogen is also popular due to its rapid crosslinking when it is inter-
changeably bioprinted with thrombin into a defect. Photo-crosslinkable bioinks, such as gelatin
methacrylamide (GelMA), hyaluronic acid methacrylate (HAMA), and poly(ethylene glycol), have
been commonly used in bioprinting [30]. Since exposing UV light directly to a live subject can

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be dangerous [31], one practical example is to expose UV light towards the side of a transparent
nozzle during bioink extrusion [32]. Recently, visible light photo-initiating systems have become
popular, which avoided the use of UV light favoring cell viability in photo-crosslinkable bioinks
[33–35]. In addition to existing bioinks, more alternatives are expected to be developed to
broaden the options for IOB. Newer materials may facilitate retention of the bioprinted constructs
at the desired site without the utilization of a support dressing/scaffold, such as a vacuum
assisted closure device. The manufacture of rapidly integrable bioinks would expand the feasi-
bility of bioprinting into enclosed cavities (e.g., abdomen, thorax), which are not conducive to graft
immobilization techniques when compared with more superficial sites, such as the skin.

Automation of IOB Processes

Imaging of defects during IOB should be performed in a minute timescale immediately after a sur-
gical excision since the prolonged exposure of the defect can increase surgical complications
[36]. To match this requirement, scanners based on 3D photogrammetry provide a supreme
solution to obtain raw data of the defect topography during IOB. Several models of portable pho-
togrammetric scanners (e.g., Artec Space Spider and CREAFORM HandySCAN) are able to
reach a resolution up to ~30 μm, with an extremely short scan time (b5 min), and the portability
offers the possibility to get the defect scanned easily. To create a model of tissue constructs,
image processing is necessary, including image preprocessing, segmentation, feature extraction,
and data mining [37]. During IOB, all these processes should be completed in a few minutes after
obtaining the scanned data. Hence, segmentation software is vital for convenient extraction of the
region of interest from 3D images [38,39]. Since defects have unique textural features case by
case, improper postprocessing of images might generate pointless 3D models, which needs to
be carefully evaluated. Artificial intelligence and robotics can be incorporated to reduce the
process time and variation caused by operators. So far, researchers have strived to optimize
path planning targeted at IOB [40,41], including printing on a free-moving hand anatomy
using motion tracking [42,43]. In the future, machine learning can be used to automatically
generate optimal bioprinting strategies [44–47]. The integration of 3D scanner with a fixed relative
coordinate to the robotic arm is also required to eliminate the need of prebioprinting calibration to
minimize total processing time.

Due to shape distortions caused by the sol–gel transition of hydrogels and movement of the live
subject during surgery (such as breathing), automation of IOB with tight control on high resolution
is also challenging. This issue might be addressed by real-time monitoring technologies to ob-
serve construct deformation and provide feedback signal for subsequent deposition [43]. Com-
mercially available bioprinters with three-axis coordinates are usually not sufficient for IOB for
irregular-shaped defects, which necessitates the use of bioprinters with higher degrees of free-
dom (DOF), where robotic arms (such as surgical robots) can be the ideal solution. Although
BioAssemblyBot with a six-axis robotic arm from Advanced Solutions Inc. (Figure 2A, Key Figure)
is currently the only commercially available bioprinter with higher DOF capability, there are many
companies specialized in robotic surgeries (e.g., Intuitive Surgical, MAKO Surgical Corp, etc.)
with the potential of integrating bioprinting capabilities into surgical robots in the future.

Vascularization in Intraoperatively Bioprinted Tissues

Vascularization is crucial for maturation of bioprinted constructs, especially in segmental defects.
Although bioprinted constructs with an embedded microvasculature have been described [4], it is
still not feasible to directly anastomose them to the recipient vasculature. Therefore, engineered
grafts still rely on inosculation from the recipient bed for perfusion. In order to drive
microvascularization within intraoperatively bioprinted tissues, there are multiple ways, such as
bioprinting of endothelial progenitor cells, hypoxia-inducible factor (HIF), or vascular endothelial

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Key Figure
Intraoperative Bioprinting (IOB) in a Surgical Setting and its Application for Repair of Composite Tissues

Trends in Biotechnology

Figure 2. Conceptual schematic diagram of (A) IOB in a surgical setting, and (B) the regeneration of composite tissues in a stratified manner. In the case where blood
vessels are retained in host tissue; they can be relocated (left) prior to the deposition of layer-specified bioinks containing different cell types (right).

growth factor (VEGF). Since it usually takes more than 10 days for angiogenesis to take place [48],
temporary strategies can be followed to extend the oxygen supply prior to inosculative angiogen-
esis. For example, oxygen-filled microparticles or oxygen-generating biomaterials (OGBs)

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can be bioprinted within the bioink, which are expected to feed the cells until capillaries can ac-
tively transfer the blood [49,50]. Oxygenation rate of OGBs (e.g., peroxides) is the key parameter
for viable outcome of organs, which can be controlled by factors such as pH, temperature, and
solubility of peroxides [51]. Involvement of porous internal structures is an advantage of
bioprinting and another option to facilitate infiltration of blood from host tissue, which offers the
possibility for IOB of porous constructs. Introduction of macropores in IOB can be realized by
bioprinting filaments in crosshatched patterns similar to what has been achieved in conventional
in vitro bioprinting [52].

However, the ultimate goal is to create a bioprinted construct that includes an embedded mi-
crovasculature with a continuous anastomosable artery and vein. With the development of
super-microsurgical techniques, it is now feasible to perform a direct anastomosis on vessels
with an internal diameter b150 μm [53]. This advancement may provide a mechanism for the
rapid establishment of graft blood flow without requisite exposure to main vascular tree. This
is significant as the vascular pedicle of the graft can be kept quite short, simplifying the
bioprinting process. To facilitate graft anastomosis and vascularization, the implanting surgeon
can also utilize a variety of autologous conduits, including the creation of arteriovenous
loops.

IOB of Composite Tissues and Their Translational Potential

Currently, seamless reconstruction of tissues with multiple components such as
craniomaxillofacial (CMF) defects (skin, bone, and muscle), osteochondral defects (car-
tilage and bone), and musculoskeletal defects (bone, muscle, tendon, and skin) possesses
several limitations. Different tissue types exhibit local variations in terms of physiological, an-
atomical, and histological aspects and precise layer-by-layer stacking of multiple tissue com-
partments is not trivial. Such compartmentalization necessitates the precision and effective
use of stem cells and differentiation factors, since differentiating stem cells into multiple lin-
eages is crucial in order to recapitulate the native tissue anatomy. Taking a major musculo-
skeletal defect as an example, it comprises layers of bone, blood vessel, muscle,
subcutaneous fat, and skin tissues from inside out, which contains more than 10 cell types
(Figure 2B) and has traditionally required an autologous composite fibula free-flap for
correction.

IOB of composite tissue should allow rapid acquirement of the defect information with mini-
mum manual interventions, enabling personalized reconstructions in an anatomically accurate
and cosmetically appealing manner immediately after characterization of the defect. Although
bioprinted constructs are usually designed based on the compartmentalization of native tis-
sues, the maturation of bioprinted tissues may alter its anatomy and phenotype, leading to dif-
ferences from the native tissue. Since composite tissues are usually thick, vascularization is
particularly vital to enhance the proper tissue regeneration, such as bone formation in CMF de-
fects, as necrotic scar tissue would take over if vascularization is not sufficient [54]. Currently,
free-flap surgery is the standard of care for the repair of composite segmental defects. A free-
flap is an autologous tissue transplant where expendable donor tissue along with its feeding
vascular pedicle (artery and vein) is transferred to a remote recipient site [55]. This approach
has revolutionized the treatment of large traumatic and oncologic defects. However, free-flap
surgery can be restricted by limited donor site supply and donor site morbidity. Additionally, al-
though the goal is to replace like tissue with like tissue, this is often impossible. For example, a
fibula free-flap can be used for intra-oral bone and soft tissue replacement in mandible recon-
struction but the amount of bone stock is reduced, sensation is lost, and skin is not equivalent
to native mucosa. Therefore, it may be better to combine the principles of reconstructive

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microsurgery and IOB. The surgeon could configure the recipient vasculature to perfuse an ad- Outstanding Questions
jacent bioprinted construct either via direct anastomosis or angiogenic induction. This would What are some efficient methods
eliminate the concerns of donor supply and morbidity while providing an exact match of the de- to facilitate vascularization and
microcapillarization in an intraoperatively
sired replacement tissue. In the future, it is anticipated that bioprinted or tissue engineered
bioprinted construct?
grafts will be available instead of harvesting autologous flaps.
How can bioprinting modalities be
Another concern about IOB of composite tissues is the integration of bioprinted tissues to native integrated and miniaturized for
intraoperative bioprinting of composite
tissue, especially for avascular tissues, such as cartilage due to its low metabolism and anti-
tissues?
adhesive ECM [56]. Vertical integration of cartilage to underlying bone occurs due to the innate
repair capability of bone [56]. In order to enhance the healing capability, intraoperatively bioprinted How can image processing, robotics
composite tissues with a histologically similar osteochondral interface will be a promising solution and artificial intelligence contribute
to the automation of intraoperative
[2], which can be integrated into full-thickness bone-cartilage defects. However, lateral integration bioprinting technologies?
of the bioprinted cartilage to the adjacent cartilage is a major roadblock to achieve permanent
cartilage replacement [56]. Strategies to enhance lateral integration may include bioink How can current surgical methods be
combined with intraoperative bioprinting
functionalization to enable direct bonding to the adjacent cartilage [57].
to promote its clinical translation,
especially in the context of repairing of
So far, animal models for IOB are almost non-load-bearing, such as skin and calvarial defects composite tissues?
(Table 1). IOB for repairing load-bearing defect models, such as segmental bone defects in
Beyond the scientific barriers, how can
long bones and joint defects, will gain more attention in the future. Mechanical stiffness of intraop- ethical, regulatory, and intellectual
eratively bioprinted constructs depends on their inherent properties determined by the bioink, property issues be addressed for
which can be resolved by developing new materials such as tough hydrogels [58] or integrating successful clinical translation of
mechanically strong thermoplastics. In this regard, bioprinting of soft bioinks can be coupled intraoperative bioprinting technology?

with thermoplastics [e.g., polycaprolactone (PCL) or poly(lactic-co-glycolic acid) (PLGA)], where

one can envision a technology that can facilitate rapid cooling of deposited thermoplastics into
a defect in a safe manner (such as laser-based cooling systems [59]). In addition, postoperative
care and rehabilitation are still necessary (as in conventional surgeries) until intraoperatively
bioprinted tissue restores sufficient mechanical strength.

Concluding Remarks
IOB has already shown promise for regeneration of tissues, including cartilage, skin, and bone,
in animals. However, regeneration of composite tissues, which are composed of hard and
soft tissues, and the interface layers in between, have hardly been explored, requiring the devel-
opment of new bioinks with rapid and stable crosslinking and the integration of advanced
bioprinting modalities.

In addition, seeking a practical way to facilitate and enhance vascularization is vital for long-term
functionality of intraoperatively bioprinted tissues. It is especially appealing to combine IOB with
vascular anastomosis to repair composite segmental defects, which is currently treated by
free-flap surgeries. In the long term, automation of IOB not only relies on the integration of se-
quential processes, but also requires a large amount of clinical cases to optimize the bioprinting
strategies (see Outstanding Questions). Beyond scientific considerations, clinical studies will be
impeded by ethical and regulatory issues and sharing of patient-specific information for database
development will be related to the protection of private information and intellectual properties.
Although significant efforts will be required to address all these issues, we do not doubt that
IOB will be a game changer in regenerative surgical care.

Acknowledgments

This research was funded by the Osteology Foundation (15-042, 18-076), International Team for Implantology (1275_2017),
National Science Foundation (1600118), and National Institutes of Health (1R01DE028614-01A1). The authors are also
thankful to Advanced Solutions Inc. and Ms Talley Fisher (from Penn State) for providing Figure 2A and 2B, respectively.

10 Trends in Biotechnology, Month 2020, Vol. xx, No. xx

Trends in Biotechnology
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